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| Status |
Public on Jan 31, 2018 |
| Title |
TRAF6 Mediates Basal Activation of NF-κB Necessary for Hematopoietic Stem Cell Homeostasis. |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The nuclear factor-kB (NF-kB) family of transcription factors is important for hematopoietic function, including development, maintenance, and differentiation of different hematopoietic lineages in response to cytokines and infection. Although ligand-independent or basal NF-kB signaling is required for HSC homeostasis in the absence of inflammation, the upstream tonic mediators of NF-kB signaling are not known. Herein we describe TNF receptor associated factor 6 (TRAF6) as an essential regulator of HSC homeostasis by preserving self-renewal and quiescence through basal activation of NF-kB. Hematopoietic-specific deletion of Traf6 resulted in impaired HSC self-renewal and fitness. Gene expression, RNA splicing, and molecular analyses of Traf6-deficient HSPC revealed changes in adaptive immune signaling, innate immune signaling, and NF-kB signaling, indicating that signaling via TRAF6 in the absence of cytokine stimulation and/or infection occurs in HSPC and is required for HSC function. In addition, we established that loss of NF-kB signaling is responsible for the major hematopoietic defects observed in Traf6-deficient HSPC as deletion of IKKb similarly resulted in impaired HSC self-renewal and fitness. Taken together, our observations position TRAF6 as an essential regulator of HSC homeostasis by maintaining a minimal threshold level of IKKb/NF-kB signaling.
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| Overall design |
Hematopoietic stem and progenitor cell (HSPC)-enriched lineage-Sca1+Kit+ (LSK) cells were sorted from mice reconstituted with Vav-Cre+ and Traf6-/-;Vav-Cre+ bone marrow cells. Total RNA was extracted from LSK cells and purified with Quick-RNA MiniPrep Kit (Zymogen). RNA quality was determined using the Agilent Bioanalyzer 2100 (Hewelett Packard). 100 ng total RNA was used for enrichment of poly A RNA with the Apollo 324 (WaferGen, Fremont, CA). Poly A RNA was further fragmented (RNase III), adaptor-ligated, and reverse transcribed (Superscript III reverse transcriptase, Lifetech, Grand Island, NY), followed by purification using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis IN). To prepare libraries, universal and index-specific primers, and sample-specific index were added to each adaptor-ligated cDNA sample to amplify the library, followed by purification using AMPure XP beads. The quality and yield of the libraries were assessed on the Bioanalyzer (Agilent, Santa Clara, CA). Libraries at the final concentration of 15 pM were clustered onto a PE flow cell using Illumina's TruSeq PE Cluster kit v3, and sequenced using TruSeq SBS kit on the Illumina HiSeq system.
To study differential gene expression, individually indexed libraries were proportionally pooled (20-50 million reads per sample in general) for clustering in cBot system (Illumina, San Diego, CA). Libraries at the final concentration of 15 pM were clustered onto a single read (SR) flow cell using Illumina’s TruSeq SR Cluster kit v3, and sequenced for 50 bp using TruSeq SBS kit on Illumina HiSeq system.
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| Contributor(s) |
Fang J, Muto T, Kleppe M, Bolanos LC, Hueneman KM, Walker CS, Sampson L, Wellendorf AM, Chetal K, Choi K, Salomonis N, Choi Y, Zheng Y, Cancelas JA, Levine RL, Starczynowski DT |
| Citation(s) |
29386112 |
| Submission date |
Jan 10, 2018 |
| Last update date |
Feb 25, 2019 |
| Contact name |
Jing Fang |
| Organization name |
University of South Carolina
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| Department |
Drug Discovery and Biomedical Sciences
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| Street address |
715 Sumter Street
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| City |
Columbia |
| State/province |
SC |
| ZIP/Postal code |
29208 |
| Country |
USA |
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| Platforms (1) |
| GPL15103 |
Illumina HiSeq 1000 (Mus musculus) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA429479 |
| SRA |
SRP128739 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE109050_RAW.tar |
1.1 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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